Prevention of Herpes Keratitis by Monoclonal Antibodies Specific for Discontinuous and Continuous Epitopes on Glycoprotein D

Home , Herpes simplex keratitis

Robert N. Lausch,* Herman Stoats,* John E. Oakes,* Gory H. Cohen.fi: and Roselyn J. Eisenberg:J:§

Seven monoclonal antibodies (mAb) specific for defined discontinuous and continuous epitopes on glycoprotein D of herpes simplex virus type 1 (HSV-1) were surveyed for their capacity to protect against virus-induced corneal disease in a murine ocular infection model. A known amount of purified mAb was transferred passively to BALB/c mice 24 hr after topical infection with HSV-1 on their scarified corneas. At high doses (50-136 ng), all seven mAbs protected against the development of persistent necrotizing stromal keratitis. Significant protection was also observed at low doses (20 ng) with two mAbs to discontinuous epitopes and two mAbs to continuous epitopes. Selected high-dose mAbs also were able to reduce the severity of blepharitis. These results indicated that at least seven different antigenic sites on glycoprotein D can serve as targets for effective antibody therapy in the murine model of HSV-1 ocular infection. Invest Ophthalmol Vis Sci 32:2735-2740,1991

Glycoprotein D (gD) is a major structural compo- nize distinct common and type-specific sites on HSV nent of the herpes simplex virus (HSV) envelope and type 1 (HSV-1) and HSV type 2 (HSV-2) gDs.14>15 is required for virus replication in tissue culture.1 Re- Several mAb groups recognize discontinuous epi- cent studies identified a critical site on this molecule topes that are dependent on proper tertiary structure; that is involved in virus penetration into cells.2'3 The others recognize continuous epitopes that are present gD is expressed on the surface of virus-infected cells in both the denatured and native forms of the mole- and on the virus particle; it is an important antigen, cule. inducing both cellular4 and humoral5"7 immune re- The availability of mAbs to HSV antigens stimu- sponses in humans. The potent immunogenicity of lated interest in whether they might be effective as gD led to its evaluation as a vaccine candidate. Many antiviral agents in vivo. Initially, it was shown by sev- studies established that immunization to gD, or its eral laboratories that passive transfer of mAbs to the peptides, can protect against acute infection and re- major glycoproteins could protect mice against the duce the incidence of latency and recurrent dis- development of a fatal encephalitis.16"19 Metcalf and ease. co-workers20 were the first to show that mAbs to gB, The antigenic nature of gD is complex, and it still is gC, and gD also could protect animals against corneal not defined completely. Analysis of its antigenic sites disease when passively transferred after ocular infec- was investigated principally through use of monoclo- tion. Subsequently, we conducted studies with a puri- nal antibodies (mAbs) specific for this molecule. fied anti-gD mAb and found that concentrations as These antibodies were arranged into groups that recog- low as 10 ng could protect against necrotizing stromal keratitis.21 From the *Department of Microbiology/Immunology, Univer- Previous passive immunization studies were con- sity of South Alabama, Mobile, Alabama, and the tDepartment of ducted with mAbs whose reactivity with specific anti- Microbiology and Center for Oral Health Research, ^School of Den- genic sites on the HSV glycoproteins was unknown. tal Medicine, and §Department of Pathobiology, School of Veteri- We exploited the availability of mAbs to defined dis- nary Medicine, University of Pennsylvania, Philadelphia, Pennsyl- vania. continuous and continuous epitopes on gD to investi- Supported in part by National Institutes of Health research grants gate whether certain antigenic sites on this important EY07564, DE08239, and All8289, Bethesda, Maryland, and by a molecule were more effective than others as targets grant from American Cyanamid Company, Wayne, New Jersey. for antibody therapy. Our experimental approach was Submitted for publication: November 19, 1990; accepted May to test whether known concentrations of purified 14, 1991. Reprint requests: Dr. Robert N. Lausch, Department of Microbi- mAbs to selected epitopes would be capable of pre- ology/Immunology, College of Medicine, University of South Ala- venting blinding corneal disease in mice infected topi- bama, Mobile, AL 36688. cally with HSV-1.

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Materials and Methods Corneal Infection Animals For corneal infection, the mice were anesthetized Four-week-old female BALB/c mice were obtained with 0.2 ml of a 1:10 dilution of sodium pentobarbital from Charles River Breeding Laboratories (Wilming- (50 mg/ml stock solution), and one eye was scarified ton, MA). The animals were treated and housed in by three twists of a 2-mm corneal trephine. A 2-jil accordance with the ARVO Resolution on the Use of volume containing the desired concentration of HSV- Animals in Research. 1 RE was dropped onto the corneal surface and gently massaged into the eye with the eyelids. The eyes were Virus and Cells examined two to three times a week for the first 2 weeks after infection and then weekly thereafter using The virus used for corneal infection was HSV-1 a stereo microscope with a fiberoptic light source. strain RE. It was plaque purified three times, and then Stromal keratitis and blepharitis were graded on a virus stocks were grown and titrated on Vero cells as scale of 0 (no disease) to +5 (most severe disease) as previously described.22 The HSV-1 strain KOS, used previously described.20-23 All clinical scoring was done in the neutralizing antibody tests, also was grown and in a masked fashion. The data were analyzed using the titered on Vero cells. The latter were grown in Dul- Mann-Whitney U test. becco's modified minimal essential medium containing 5% calf serum. Serologic Tests Each purified monoclonal antibody was tested for Monoclonal Antibodies its capacity to neutralize HSV-1. Neutralizing tests For this study, seven anti-gD mouse mAbs were were conducted by incubating equal volumes of di- used. These were 8D2, DL11, DL2, BD78, DL6, ID3, luted antibody with strain KOS and guinea pig com- and BD66. The epitope specificity of each is summa- plement (1:15 dilution). After 30 min incubation at rized in Table 1 and discussed subsequently. Before 37°C, the residual infectious virus was assayed on purification, each mAb-ascites fluid preparation was Vero monolayers in 24-well plates. Appropriate posi- filtered (0.22 /*m) to remove particulate matter. The tive and negative controls were included in each test. filtered samples then were diluted 1:2 with phosphate- Antibody binding to HSV-1 RE strain-infected buffered saline (PBS) and purified using an immobi- Vero cells and uninfected cells were assayed in the lized protein G column (Genex, Gaithersburg, MD) ELISA. The protocol used was that previously de- according to the manufacturer's instructions. After scribed,24 except that 0.01% poly L-lysine was used to elution from the column, the immunoglobulins were pretreat the plates. Target cells were fixed with 0.5% concentrated using a Centriprep 30 ultrafiltration cell glutaraldehyde and blocked with 1.0% bovine albu- (Amicon, Beverly, MA). The resulting concentrated min in borate-buffered saline. mAbs were dialyzed against PBS, and the protein con- tent was determined using the BCA* protein assay Results (Pierce, Rockford, IL). Immunoglobulin isotypes were determined using Specificity and Properties of Anti-GD mAbs an enzyme-linked immunosorbent assay (ELISA) The gD present in HSV-1 (gD-1) is 85% homolo- kit obtained from Southern Biotechnology (Bir- gous with gD-2 found in HSV-2.15 The former (in ma- mingham, AL). The titration end point was desig- ture form) consists of 369 amino acids; the latter has nated as that at which the optical density was three- 368 amino acids. The mAbs that recognize discon- fold higher or more than the control optical density. tinuous epitopes on gD were arranged into four groups, one of which was subdivided. We selected mAbs 8D2, DL11, and DL2 that recognize discon- Table 1. Properties of Purified Anti-gD mAbs tinuous epitopes III (RJE and GHC, unpublished ob- 25 15 26 HSV-1 servations), Ib, and VI, ' respectively, for study Antibody Type igG ELISA Neutralizing (Table 1). mAb Group Epitope Isotype Titer Titer Four additional mAbs studied recognized continu- 8D2 III Discontinuous 2a 106 103 ous epitopes (Table 1). Specifically, BD78 and DL6 DL11 Ib Discontinuous 2a 106 103 recognized overlapping epitopes at antigenic site II.27 DL2 VI Discontinuous 2b 105 <10' BD78 II Continuous 1 106 10' The BD78 binds to residues 264-275; DL6 binding DL6 II Continuous 2a 107 102 was mapped to amino acids 272-279. The MAb ID3 VII Continuous 2a 105 102 6 BD66 recently was shown to bind to amino acids BD66 XI Continuous 1 10 <10' 284-301, an antigenic site designated XI.27 Finally,

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ID3 is a group VII mAb that recognizes a continuous epitope mapped to residues 1-23.1528 125 All seven mAbs (after purification) were tested for their capacity to bind to HSV-1 RE-infected cells and neutralize HSV-1 KOS infectivity. Table 1 shows the isotype, the ELISA titer against Vero cells infected with HSV-1 strain RE, and the HSV-1 neutralization titer of each mAb. Two of the mAbs, DL2 and BD66, did not show detectable neutralizing activity. Each of 55 the antibodies recognized a type common epitope ex- 136 121 cept for DL2 which was gD-1 specific.26

Protective Activity of mAbs Specific for Discontinuous Epitopes on gD

Previous studies found that purified mAb 8D2 was protective when passively transferred in the 10-50-^g dose range.21 For our initial studies, we tested whether mAbs to discontinuous epitopes Ib and VI also could prevent the development of virus-induced stromal keratitis. Four-week-old BALB/c mice were infected on the right trephined cornea with 2 X 104 plaque- forming units of HSV-1 strain RE. Twenty-four hours Control 8O2 DL11 DL2 later, the animals were divided into groups of six to eight each and given a single intraperitoneal inocula- ANTI-{p MAB"8 USED FOR TREATMENT tion of high-dose purified mAb. The control animals Fig. 1. Capacity of mAbs specific for discontinuous epitopes on received nonimmune globulin of the immunoglobu- gD to resolve HSV-1-induced corneal disease. The number at the top of each bar indicates the micrograms of antibody passively lin G2a isotype. The mice then were followed for the transferred. The mean opacity scores 4 weeks postinfection after development of corneal disease. (A) high-dose and (B) low-dose therapy are shown. There were six Figure 1A shows the mean corneal opacity score 4 mice per group. *P < 0.025; **P < 0.005. weeks postinfection. All eight control mice had severe necrotizing stromal keratitis that persisted through the remainder of the 7-week observation period. Each Capacity of Anti-gD mAbs to Protect Against of the three mAbs to discontinuous epitopes was Blepharitis strongly protective (P < 0.005). The HSV-1 infection of the mouse eye consistently Next, the protective capacity of low-dose mAbs was induces blepharitis and corneal disease. The severity evaluated (Fig. IB). Passive transfer of 20 ng of 8D2 of eyelid disease usually peaks during the first week of was highly protective (in agreement with previous ob- infection and then gradually diminishes. The capacity servations).21 At the 20-/ig dose, DL11 resolved cor- of the passively transferred mAbs to limit the develop- neal opacity in 60% of the recipients; DL2 was not ment of this disease was examined. Figure 3 shows the protective (P > 0.05). mean blepharitis score 7 and 14 days postinfection in animals given high-dose antibody. Selected mAbs to Protective Activity of mAbs Specific for Continuous discontinuous epitopes (8D2 and DL11) and continu- Epitopes on gD ous epitopes (ID3 and DL6) were able to reduce the severity of eyelid disease significantly, but the protec- Four mAbs that recognized continuous epitopes on tive effect was relatively modest. On days 7 and 14, gD were tested for their capacity to protect against none of the mAbs given at the 20-/ug concentration stromal keratitis. Figure 2A shows that, at high anti- was protective (data not shown). Overall, the antibod- body doses, all four were strongly protective (P ies most effective in preventing or reducing the sever- < 0.005) compared with the control group. However, ity of stromal keratitis were the same as those that treatment with ID3 and DL6 mAbs appeared to be ameliorated blepharitis. more efficacious than that with BD78 and BD66 mAbs. This impression was confirmed by low-dose Discussion testing, ie, at the 20-^g test dose, only ID3 and DL6 We conducted this study to determine whether gave significant (P < 0.05) pro action (Fig. 2B). mAbs specific for discrete antigenic sites on gD could

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Previous work showed that 8D2 mAb could reduce the severity of blepharitis but not prevent the disease.23 Our study confirmed this findingan d extended it to show that both continuous and discontinuous epitopes can function as protective target antigens in cutaneous eyelid disease. The observation that protection was modest and mediated by only four of the seven mAbs tested supports the view that humoral immunity directed against gD is therapeutically more effective against corneal than HSV-induced eyelid disease. When immunoglobulin was transferred passively at low doses (20 /zg) only four of the seven mAbs protected significantly against corneal opacity. The rea- son for this is unclear. It may be more than a coinci- dence that all four protective mAbs were of the immunoglobulin G2a isotype; the three nonprotective antibodies had a different isotype. Studies by others found that immunoglobulin G2a antibodies are syn- thesized preferentially by mice after viral infection,32 and of the four murine immunoglobulin G isotypes, G2a is the most efficient at mediating antibody-de- 3334 Control ID3 DL6 BD78 BD66 pendent cellular cytotoxicity. It is also possible

ANTI-tf) MAffs USED FOR

Fig. 2. Capacity of mAbs specific for continuous epitopes on gD Day 7 to resolve HSV-1 -induced corneal disease. The number at the top of each bar indicates the micrograms of antibody passively trans- 54 ferred. The mean opacity scores 4 weeks postinfection after (A) 125 121 50 high-dose and (B) low-dose therapy are shown. There were six mice per group. *P < 0.05; **P < 0.005.

protect against the development of HSV-1-induced stromal keratitis. Antibodies specific for six distinct regions on the molecule were tested, and it was found that each could protect if a sufficient concentration of immunoglobulin was administered. Thus, both con- Day 14 tinuous epitopes II, VII, and XI and discontinuous epitopes Ib, III, and VI could serve as effective targets 29 54 on gD. Others previously reported that a mAb spe- 125 cific for antigenic site Ib would protect mice against 50 intracranial challenge with HSV-1. The mAbs reac- tive with site la were shown to protect against lethal footpad challenge with HSV-1 or HSV-216 and against zosteriform spread of HSV-1 in the flanks of mice.30 In our study, the protective effect of high-dose antibody was independent of the antibody isotype; immu- Control 8O2 DL11 DL2 ID3 0L6 BO78 BO66 noglobulin G2a, G2b, and G1 antibodies all were ther- ANTI-tf) MABs USED FOR TREATV&TT apeutically active. Also, mAbs DL2 and BD66 (which did not display any detectable neutralizing activity Fig. 3. Capacity of mAbs specific for discontinuous and continuous epitopes on gD to reduce the severity of HSV-1 -induced blepha- against HSV-1) were able to prevent stromal disease. ritis. The experimental details are the same as those in Figures 1A The absence of a correlation between neutralizing an- and 2A. The mean blepharitis scores for day 7 (A) and day 14 (B) tibody titers and levels of protection also was ob- postinfection are shown. There were six mice per group. *P < 0.025; served in other studies with HSV.1718-2931 **P< 0.01; ***P< 0.005.

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that the affinities of the protective antibodies were References higher than those of the nonprotective mAbs. This 1. Ligas MW and Johnson DC: A herpes simplex virus mutant in could explain why DL6 and BD78, which recognize which glycoprotein D sequences are replaced by /3-galactosi- overlapping epitopes, differ in their therapeutic po- dase sequences binds to but is unable to penetrate into cells. J tential. Virol 62:1486, 1988. It is not known how a single intraperitoneal inocula- 2. Feenstra V, Hodaie M, and Johnson DC: Deletions in herpes tion of mAb at doses as low as 10-20 ng is able to simplex virus glycoprotein D define nonessential and essential domains. J Virol 64:2096, 1990. prevent corneal blindness. The host immune re- 3. Muggeridge MI, Wilcox WC, Cohen GH, and Eisenberg RJ: sponse is thought to play an important role in the Identification of a site on herpes simplex virus type 1 glycopro- development of HSV-induced stromal keratitis.35"37 tein D that is essential for infectivity. J Virol 64:3617, 1990. Previous histologic studies found that, in contrast to 4. Zarling JM, Moran PA, Lasky LA, and Moss B: Herpes sim- untreated infected corneas, there is little or no mono- plex virus (HSV)-specific human T-cell clones recognize HSV glycoprotein D expressed by a recombinant vaccinia virus. J nuclear cell infiltrate of the corneas of antibody- 21 Virol 59:506, 1986. treated hosts. Thus, we originally postulated that an- 5. Vestergaard BF: Quantitative immunoelectrophoretic analysis tibody might protect by suppressing the host immune of human antibodies against herpes simplex virus antigens. In- response. However, this explanation is not possible fect Immun 23:553, 1979. because additional studies showed that immunoglobu- 6. Gilman SC, Docherty JJ, and Rawls WE: Antibody responses in human to individual proteins of herpes simplex viruses. In- lin treatment does not inhibit antibody formation, fect Immun 34:880, 1981. and it accelerates the development of delayed hyper- 7. Zweerink HJ and Stanton LW: Immune response to herpes 23 sensitivity. simplex virus infections: Virus-specific antibodies in sera from In our model, antibody treatment did not enhance patients with recurrent facial infection. Infect Immun 31:624, significantly infectious virus clearance from the eye.21 1981. 8. Chan WL: Protective immunization of mice with specific Thus, it seems unlikely that the passively transferred HSV-1 glycoproteins. Immunology 49:343, 1983. immunoglobulin protected primarily by promoting 9. Lasky LA, Dowbenko D, Simonsen CC, and Berman PW: Pro- lysis of virus infected cells. Others38 also found that tection of mice from lethal herpes simplex virus infection by passively transferred antibody did not reduce HSV vaccination with a secreted form of cloned glycoprotein D. Bio- titers at the inoculum site, even when the antibody technology 2:527, 1984. 10. Long D, Madara TJ, Ponce de Leon M, Cohen GH, Mont- was given 1 day before corneal infection. Their pro- gomery PC, and Eisenberg RJ: Glycoprotein D protects mice vocative hypothesis is that the antibody protects against lethal challenge with herpes simplex virus types 1 and 2. against severe ocular damage by restricting virus Infect Immun 43:761, 1984. spread from the nervous system back into the eye. 11. Eisenberg RJ, Cerini CP, Heilman CJ, Joseph AD, Dietzschold Whatever the correct explanation, it is likely that B, Golub E, Long D, Ponce de Leon M, and Cohen GH: Syn- thetic glycoprotein D-related peptides protect mice against these antibodies exert their therapeutic effect through herpes simplex virus challenge. J Virol 56:1014, 1985. collaboration with other antiviral defense mecha- 12. Foster CS, Sandstrom IK, Wells PA, Thompson P, Daigle J, nisms. and Opremcak EM: Immunomodulation of experimental mu- In summary, antibodies specific for seven different rine herpes simplex keratitis: II. Glycoprotein D protection. antigenic sites widely distributed on the gD molecule CurrEyeRes7:1051, 1988. 13. Inoue Y, Ohashi Y, Shimomura Y, Manabe R, Yamada M, could protect against HSV-1-induced corneal blind- Ueda S, and Kato S: Herpes simplex virus glycoprotein D: Pro- ness. Our results indicate that humoral immunity can tective immunity against murine herpetic keratitis. Invest Oph- facilitate resolution of HSV infection and minimize thalmol Vis Sci 31:411, 1990. cellular infiltration of the cornea. Thus, these findings 14. Eisenberg RJ, Long D, Pereira L, Hampar B, Zweig M, and provide a rational basis for investigating whether Cohen GH: Effect of monoclonal antibodies on limited proteo- lysis of native glycoprotein gD of herpes simplex virus type I. J monoclonal or polyclonal antibodies would be useful Virol 41:478, 1982. in the treatment of HSV corneal infection in humans. 15. Muggeridge MI, Roberts SR, Isola VJ, Cohen GH, and Eisen- berg RJ: Herpes simplex virus. In Immunochemistry of Vi- Key words: herpes simplex virus, monoclonal antibodies, ruses, Vol 2, van Regenmortel MHV and Neurath AR, editors. Amsterdam, Elsevier, 1990, pp. 459-481. stromal keratitis, glycoprotein D, blepharitis 16. 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herpes simplex virus type 1 infection. Infect Immun 38:168, E, Varrichio A, Pereira L, and Eisenberg RJ: Localization and 1982. synthesis of an antigenic determinant of herpes simplex virus 19. Kino Y, Eto T, Ohtomo N, Hayashi Y, Yamamoto M, and glycoprotein D that stimulates production of neutralizing anti- Mori R: Passive immunization of mice with monoclonal antibody. J Virol 49:102, 1984. bodies to glycoprotein gB of herpes simplex virus. Microbiol 29. Kumel G, Kaerner HC, Levine M, Schroder CH, and Glorioso Immunol 29:143, 1985. JC: Passive immune protection by herpes simplex virus-spe- 20. Metcalf, JF, Koga J, Chatterjee S, and Whitley RJ: Passive cific monoclonal antibodies and monoclonal antibody-resis- immunization with monoclonal antibodies against herpes sim- tant mutants altered in pathogenicity. J Virol 56:930, 1985. plex virus glycoproteins protects mice against heipetic ocular 30. Simmons A and Nash AA: Role of antibody in primary and disease. Curr Eye Res 6:173, 1987. recurrent herpes simplex virus infection. J Virol 53:944, 1985. 21. Lausch RN, Oakes JE, Metcalf JF, Scimeca JM, Smith LA, and 31. Rector JT, Lausch RN, and Oakes JE: Identification of in- Robertson SM: Quantitation of purified monoclonal antibody fected cell-specific monoclonal antibodies and their role in host needed to prevent HSV-1 induced stromal keratitis in mice. resistance to ocular herpes simplex virus type 1 infection. J Gen Curr Eye Res 8:499, 1989. Virol 65:657, 1984. 22. Lausch RN, Kleinschrodt WR, Monteiro C, Kayes SG, and 32. Coutelier JP, Van Der Logt JTM, Heessen FWA, Warnier G, Oakes JE: Resolution of HSV corneal infection in the absence and Van Snick J: IgG2a restriction of murine antibodies elic- of delayed-type hypersensitivity. Invest Ophthalmol Vis Sci ited by viral infections. J Exp Med 165:64, 1987. 26:1509, 1985. 33. Herlyn D and Koprowski H: IgG2a monoclonal antibodies in- 23. Lausch RN, Staats H, Metcalf JF, and Oakes JE: Effective anti- hibit human tumor growth through interaction with effector body therapy in herpes simplex virus ocular infection: Charac- cells. Proc Natl Acad Sci U S A 79:4761, 1982. terization of recipient immune response. Intervirology 31:159, 34. Kaminski MS, Kitamura K, Maloney DC, Campbell MJ, and 1990. + Levy R: Importance of antibody isotype in monoclonal anti- 24. Oakes JE, Rector J, and Lausch RN: Lyt-1 T cells participate idiotype therapy of a murine B cell lymphoma: A study of in recovery from ocular herpes simplex virus type 1 infection. hybridoma class switch variants. J Immunol 136:1123, 1986. Invest Ophthalmol Vis Sci 25:188, 1984. 35. Metcalf JF, Hamilton DS, and Reichert RW: Herpetic keratitis 25. Muggeridge MI, Isola VJ, Byrn RA, Tucker TJ, Minson AC, in athymic (nude) mice. Infect Immun 26:1164, 1979. Glorioso JC, Cohen GH, and Eisenberg RJ: Antigenic analysis of a major neutralization site of herpes simplex virus glycopro- 36. Newell CK, Martin S, Sendele D, Mercadal CM, and Rouse tein D, using deletion mutants and monoclonal antibody-resis- BT: Herpes simplex virus-induced stromal keratitis: Role of tant mutants. J Virol 62:3274, 1988. T-lymphocyte subsets in immunopathology. J Virol 63:769, 26. Cohen GH, Isola VJ, Kuhns J, Berman PW, and Eisenberg RJ: 1989. Localization of discontinuous epitopes of herpes simplex virus 37. Hendricks RL, Tao MSP, and Glorioso JC: Alterations in the glycoprotein D: Use of a nondenaturing ("native" gel) system antigenic structure of two major HSV-1 glycoproteins, gC and of polyacrylamide gel electrophoresis coupled with western gB, influence immune regulation and susceptibility to murine blotting. J Virol 60:157, 1986. herpes keratitis. J Immunol 142:263, 1989. 27. Isola VJ, Eisenberg RJ, Siebert GR, Heilman CJ, Wilcox WC, 38. Shimeld C, Hill TJ, Blyth WA, and Easty DL: Passive immuni- and Cohen GH: Fine mapping of antigenic site II of herpes zation protects the mouse eye from damage after herpes sim- simplex virus glycoprotein D. J Virol 63:2325, 1989. plex virus infection by limiting spread of virus in the nervous 28. Cohen GH, Dietzschold B, Ponce de Leon M, Long D, Golub system. J Gen Virol 71:681, 1990.

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